Nucleic acids encoding neutralizing gdf8 epitope-based peptides and fusion proteins

ABSTRACT

The invention provides new, specific antigenic peptides from the protein GDF8. The invention also provides fusion proteins comprising the new peptides, immunogens and vaccines based on the new peptides and/or fusion proteins, antibodies that specifically bind to the new peptides of GDF8, and methods of treating animals in order to modulate the activity of GDF8, employing vaccines or antibodies according to the invention.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a non-provisional application that claims priorityunder 35 U.S.C. § 119(e) of provisional application U.S. Ser. No.60/533,719 filed Dec. 31, 2003, the contents of which are herebyincorporated by reference in their entireties.

FIELD OF THE INVENTION

The invention relates to the protein growth differentiation factor 8,and to antigenic peptide fragments of growth differentiation factor 87and related immunogens, vaccines, and methods of treating animals inorder to modulate the activity of growth differentiation factor 8.

BACKGROUND OF THE INVENTION

Growth differentiation factor 8 is a protein that is classified with thetransforming growth factor-β (“TGF-β”) superfamily. Generally, theproteins of the TGF-β superfamily are initially expressed as precursor(a/k/a prohormone) that undergoes proteolytic cleavage at a cluster ofbasic residues about 110-140 amino acids from the precursor proteinC-terminus. In each case, the active, or mature, TGF-β species isbelieved to be a disulfide-linked dimer of the cleaved precursor proteinC-terminal regions.

Growth differentiation factor 8, hereinafter GDF8, is also art-known asGDF-8 or myostatin. The genes encoding the precursor of GDF8(hereinafter “precursor GDF8”) have been cloned from a wide range oforganisms. These include the human and murine precursor GDF8[Nestor etal., 1998, Proc, Natl. Acad. Sci. 95:14938-43; U.S. Pat. No. 5,827,733,the contents of which are hereby incorporated by reference in theirentireties]. It has also been reported that GDF8 immunoreactivity isdetectable in human skeletal muscle in both type 1 and type 2 fibers.Antibodies and assays for detecting GDF8 are described, e.g., by U.S.Pat. No. 6,096,506.

It has further been reported that GDF8 plays a role in down-regulatingor inhibiting the growth and development of skeletal muscle, asconformed by GDF8 knock-out mice (McPherron et al., 1997, Nature3387-90). For this reason, there have been previous attempts,particularly in the field of animal husbandry, to modulate GDF8 activityin animals by several means, with the goal of down-regulating GDF8activity in order to enhance the growth, and/or relative muscle mass, ofvarious food animals.

For examples U.S. Pat. No. 6,399,312 describes a precursor GDF8 genepromoter and an assay, with the proposal that the assay be used toidentify a theoretical inhibitor of that promotor. U.S. Pat. No.6,656,475 describes a method of inhibiting the effect of GDF8 on a cellby contacting the cell with a GDF8 prodomain that competes for a GDF8receptor, and reports that the C-terminus of mature GDF8 may vary. U.S.Pat. No. 6,004,937 describes the use of follistatin as a possibleantagonist of GDF8. None of these methods has resulted in any practicalapplications in the fields of animal husbandry or clinical applications(either human or veterinary).

Others have also attempted to employ antibody and vaccine technology fordownregulating GDF8 function. For instance, U.S. Pat. No. 6,369,201 [thecontents of which are hereby incorporated by reference in theirentireties], describes peptides, i.e., fragments of GDF8 protein, and avaccine for eliciting anti-GDF8 antibodies. That patent also reported anunspecified degree of growth or weight gain, relative to controls, inrodents immunized with several of the reported GDF8 peptide fragments.

Other physiological roles for GDF8 have also been described. Forexample, U.S. Pat. No. 6,368,597, [the contents of which are herebyincorporated by reference in their entireties] has suggested thatinhibiting GDF8 function is useful for treating Type II diabetes, e.g.by administering an anti-GDF8 antibody or anti-GDF8 vaccine to a patienthaving this condition.

Nevertheless, there remains a longstanding need in the art for improvedantigens and immunogens for eliciting an anti-GDF8 immune response, aswell as for improved GDF8 antibodies capable of highly specific bindingto GDF8.

The citation of any reference herein should not be construed as anadmission that such reference is available as “Prior Art” to the instantapplication.

SUMMARY OF THE INVENTION

The present invention solves these and other shortcomings in the art byproviding GDF8 peptides (e.g., peptide fragments of GDF8 of 50 residuesor less) comprising a specific neutralizing epitope for GDF8.

In one embodiment of the invention, GDF8 peptides are provided thatinclude, for example, an isolated peptide that comprises from aboutresidue 312 to about residue 361 of natural, human precursor GDF8 (SEQID NO:1), Preferably, the inventive GDF8 peptide comprises from aboutresidue 320 to about residue 350, more preferably comprises from aboutresidue 321 to about residue 346 and most preferably comprises fromabout residue 327 to about residue 346 of natural, human precursor GDF8.The exemplified GDF8 peptide, labeled as DJ5 hereinbelow, is illustratedas follows, in both single and triple letter code, along with residuenumbering based on the precursor GDF8 of SEQ ID NO:1, for convenience.

DJ5 (SEQ ID NO:8) 327 328 329 330 331 332 333 334 335 336 337 338V   H   Q   A   N   P   R   G   S   A   G   P Val His Gln Ala Asn ProArg Gly Ser Ala Gly Pro 339 340 341 342 343 344 345 346C   C   T   P   T   K   M   S Cys Cys Thr Pro Thr Lys Met Ser

In a further embodiment of the invention, the GDF8 peptide optionallyincludes conservative single amino acid substitutions. Simply by way ofexample, these can be from one through at least five amino acidpositions within the peptide. In a particular embodiment, there is atleast one conservative amino acid substitution, e.g., between residues327 to 346 of GDF8. In another embodiment, the GDF8 peptide includesconservative amino acid substitutions at no more than five amino acidpositions within the peptide. In still another embodiment there are twoconservative amino acid substitutions between residues 327 to 346 ofGDF8. In yet another embodiment, there are three conservative amino acidsubstitutions between residues 327 to 346 of GDF8. In still anotherembodiment, there are tour conservative amino acid substitutions betweenresidues 327 to 346 of GDF8.

Preferably, the amino acid residue substitutions are at one or morepositions, relative to natural, human precursor GDF8 (SEQ ID NO: 1) thatare marked by the amino acid variations of the interspecies alignment ofFIG. 2. These are at residues 328, 329, 331, 333 and 335, andcombinations thereof, wherein,

(a) amino acid residue 328 is His, Leu or Asn (b) amino acid residue 329is Gin or Lys;

(c) amino acid residue 331 is Asn or Ser;

(d) amino acid residue 333 is Arg or Lys; and/or

(e) amino acid residue 335 is Ser, Pro or Thr.

Preferably, such modified GDF8 peptides comprise a specificneutralization epitope for an anti-GDF8 antibody and therefore retainthe property of specifically binding to an anti-GDF8 antibody, where theantibody is mAb 788 and/or an IgG fraction of goat anti-GDF8 polyclonalantiserum, as exemplified hereinbelow.

In still a further embodiment of the invention, nucleic acid molecules,i.e., polynucleotides, encoding the above-mentioned GDF8 peptides areprovided.

Preferably, the nucleic acid molecules include a section of thenaturally occurring human precursor GDF8 gene, from about nucleotide1112 to about nucleotide 1171 of the (Genebank accession NM_(—)005259,human GDF8 gene; SEQ ID NO: 2). This section encodes peptide DJ5, asdescribed above. Note that the NM_(—)005259 record includes a largeamount of sequence flanking the actual coding region. The artisan willalso appreciate that the DJ5 corresponds to nucleotides 979-1038 of theactual coding region of the GDF8 prohormone.

Replicable cloning vectors, and prokaryotic or eukaryotic host cellscomprising the nucleic acid molecules are also provided, along withmethods of producing a GDF8 peptide that include the steps of: culturingthe host cell(s), expressing the encoded peptide, and recovering thepeptide. The artisan will also appreciate that the inventive GDF8peptide will also be readily produced by any standard, art-knownchemical synthetic method.

In yet a further embodiment of the invention, a vaccine composition thatcomprises the inventive GDF8 peptide (or fusion protein thereof) is alsoprovided, e.g., that preferably includes one or more adjuvants and otherart-standard elements of a peptide or protein-based vaccine composition,Methods of eliciting an anti-GDF8 immune response in an animal,comprising administering to the animal an effective amount of thevaccine composition, are also provided.

In another further embodiment, a screening method is provided forselecting an anti-GDF8 antibody or antibody fragment from among aplurality of antibodies or antibody fragments, comprising contacting thepeptide with one or a plurality of antibodies or antibody fragments, anddetecting antibody or antibody fragment that selectively binds to thepeptide.

In yet another aspect, the present invention provides a method ofdown-regulating GDF8 activity in an animal. In one such embodiment, themethod comprises administering an antibody or antibody fragment to theanimal, in an amount and for a duration effective to down-regulate GDF8activity in the animal, wherein the antibody binds specifically to thepeptide, or by immunizing the animal with a vaccine composition asdescribed herein. The animal is preferably a vertebrate, and morepreferably a mammal, avian or fish. Preferably, the mammal is adomesticated animal (e.g., one used in animal husbandry, oralternatively, a companion animal) but can optionally be a human in needof such GDF8 downregulation.

The invention also contemplates fusion proteins incorporating theinventive GDF8 peptides. The fusion proteins can include domains thatare signal peptides, for enhanced secretion or cell surface expressionof the GDF8 fusion protein and/or to permit purification with aselective binding system. Further, the fusion proteins are contemplatedto link one or more GDF8 peptides in a single carrier protein in orderto enhance immunogenicity of the GDF8 peptide domain. In a particularembodiment, a fusion protein of the present invention comprises a GDF8peptide consisting of 50 or fewer amino acid residues that comprisesamino acid residues 327 to 346 of SEQ ID NO:1. In a related embodimentof this type, the fusion protein comprises an antigenic subfragment ofthat GDF8 peptide, e.g., a GDF8 peptide comprising about 10 consecutiveamino acid residues from DJ5 (SEQ ID NO:8).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates overlapping peptides DJ1 through DJ7, in the GDF8active region (i.e., mature GDF8), that is from residues 266-375 of theprecursor GDF8 sequence.

FIG. 2 illustrates the alignment of the human DJ5 peptide sequence (SEQID NO: 8) compared to the analogous 20-residue peptides, as located inthe precursor GDF8 proteins of the recited additional animal species.The amino acid residue positions of 321 through 347 are based on thehuman precursor GDF8. The Genebank accession numbers (incorporated byreference herein) identify the entire published protein sequence forthat species.

The aligned peptides have the following SEQ ID NOs.

Anas platyrhynchos (duck) AAL35275 (SEQ ID NO: 11) Anser anser (goose)AAL35276 (SEQ ID NO: 12) Anser anser (goose) AAR18246 (SEQ ID NO: 13)Bos taurus (cow) AAB86687 (SEQ ID NO: 14) Canis familiaris (dog)AAR14343 (SEQ ID NO: 15) Capra hircus (goat) AAR12161 (SEQ ID NO: 16)Columba livia (pigeon)AAL35277 (SEQ ID NO: 17) Coturnix chinensis(quail) AAL35278 (SEQ ID NO: 18) Danio rerio (zebrafish)AAB86693 (SEQ IDNO: 19) Equus caballus (horse) BAB16046 (SEQ ID NO: 20) Gallus gallus(chicken) AAK18000 (SEQ ID NO: 21) Gallus gallus (chicken) AAR18244 (SEQID NO: 22) Homo sapiens (human) NP-005250 (SEQ ID NO: 8) I. punctatus(catfish)AAK84666 (SEQ ID NO: 23) Lepus capensis (hare)AAN87890 (SEQ IDNO: 24) Macaca fascicularis (monkey) AAL17640 (SEQ ID NO: 25) Meleagrisgallopavo (turkey)AAB86692 (SEQ ID NO: 26) Morone chrysops (whitebass)AAK28707 (SEQ ID NO: 27) Mus musculus (house mouse)AAC531 67 (SEQID NO: 28) O. mykiss (trout) AAK71 707 (SEQ ID NO: 29) Ovis aries(sheep)AAB86689 (SEQ ID NO: 30) Papio hamadryas (baboon)AAB86686 (SEQ IDNO: 31) Rattus norvegicus (rat)AAB86691 (SEQ ID NO: 32) Salmo salar(salmon) CAC1 9541 (SEQ ID NO: 33) Sparus aurata (seabream)AAL05943 (SEQID NO: 34) Sus scrofa (pig)AAC08035 (SEQ ID NO: 35) Sus scrofa (pig)AAR18245 (SEQ ID NO: 36)

DETAILED DESCRIPTION OF THE INVENTION

Accordingly, the present invention identifies GDF8 domains that serve asa specific neutralization epitope for a polyclonal anti-GDF8 goatantiserum and for other certain specific anti-GDF8 antibodies. Theseepitopes also serve to provide fragments of the GDF8 protein that areuseful for eliciting an active and specific immune response against GDF8proteins, both in vitro, e.g. for detection of GDF8 protein, and invivo, for downregulating GDF8 activity. These fragments are generallyreferred to herein as GDF8 peptides or peptide fragments. The utility ofthese GDF8 peptides includes use as immunogens for eliciting ananti-GDF8 immune response in animals, and use as highly specificantibody-binding targets in GDF83-related assays.

The specific binding epitopes of GDF8 were identified by contactinganti-GDF8 antiserum with a battery of overlapping GDF8 peptides, anddetermining the degree of binding activity between the peptides and theantiserum IgG antibodies. The anti-GDF8 antiserum was obtained from agoat immunized with a precursor GDF8 protein having a structureoptimized for expression and antigenicity.

In order to more fully appreciate the instant invention, the followingdefinitions are provided. The use of singular terms for convenience indescription is in no way intended to be so limiting. Thus, for example,reference to a composition comprising “polypeptide” includes referenceto one or more of such polypeptides.

As used herein the term “approximately” is used interchangeably with theterm “about” and signifies that a value is within twenty percent of theindicated value i.e., a peptide containing “approximately” 50 amino acidresidues can contain between 40 and 60 amino acid residues.

It is also to be understood that this invention is not limited to theparticular configurations, process steps, and materials disclosed hereinas such configurations, process steps, and materials may vary somewhat.It is also to be understood that the terminology employed herein is usedfor the purpose of describing particular embodiments only and is notintended to be limiting, since the scope of the present invention willbe limited only by the appended claims and equivalents thereof.

As used herein, the term, “polypeptide” is used interchangeably with theterm “protein” and denotes a polymer comprising two or more amino acidsconnected by peptide bonds. Preferably, unless otherwise stated herein,the term polypeptide is distinguished from the term, “peptide” asemployed herein, by size or chain length, wherein a “peptide” refers toa polymer chain of about fifty or fewer amino acids, and a polypeptideor protein refers to polymer chain comprising more than about fiftyamino acids. Optionally, a peptide or a polypeptide may lack certainamino acid residues that are encoded by a gene or by an mRNA. Forexample, a gene or mRNA molecule may encode a sequence of amino acidresidues on the N-terminus of a polypeptide (i.e., a signal sequence)that is cleaved from, and therefore, may not be part of the finalprotein.

A “GDF8 peptide” according to the present invention is a relativelyshort fragment derived from the GDF8 protein. Even subfragments of suchpeptides can be termed GDF8 peptides. While not intending to limit themaximum size of a GDF8 peptide according to the invention, it ispreferred that the maximum size of the peptide is about 50 residues,more preferably the maximum size is about 40 residues, even morepreferably the maximum size is about 30 residues, and still morepreferably the maximum size is about 25 residues. More generally, theGDF8 peptide preferably ranges in size from about 10 to about 50 aminoacid residues in length, more preferably from about 15 to about 30 aminoacid residues, and in particular, is about 20 amino acid residues inlength. GDF8 peptides that are smaller subfragments of other GDF8peptides of the present invention, for example, a GDF8 peptidecomprising about 10 consecutive amino acid residues from DJ5 (SEQ IDNO:8), preferably comprise an antigenic portion (e.g., an epitope) fromthe larger GDF8 peptide.

In a particular embodiment, a GDF8 peptide comprises a peptide domainthat has a degree of homology ranging from about 50% to 100% homology toa peptide defined by residue numbers 327-346 (SEQ ID NO: 8) of thenaturally occurring human precursor of GDF8 (SEQ ID NO: 1). Thevariations in homology noted above are preferably conservativesubstitutions and/or variations that retain the inventive antigenicstructure that is specifically recognized by certain specific anti-GDF8antibodies. These conserved substitutions represent residuesubstitutions that are shown by interspecies homology comparisons (e.g.,see FIG. 2) to preserve GDF8 function and/or represent residuesubstitutions between amino acids of analogous chemical (e.g., physical)and electronic structure, thus preserving and/or optimizing theantibody-binding specificity of the inventive GDF8 peptides. Examples ofsuch conservative amino acid substitutions include: replacing onehydrophobic residue such as isoleucine, valine, leucine or methioninefor another; or replacing one polar residue of equivalent charge foranother, e.g., substituting arginine for lysine, glutamic acid foraspartic acid, or glutamine for asparagine.

In particular, the GDF8 peptide according to the invention will alsoinclude a specific neutralization epitope for an anti-GDF8 antibody,i.e. an epitope or antigenic domain that will specifically bind to thePGA anti-GDF8 IgG polyclonal antibody described by the Examples below,and/or that binds specifically to commercially available rat monoclonalantibody (“mAb”) Cat. No. MAB788 (R&D Systems Inc., Minneapolis. MN).

The terms “purified” or “isolated” as employed herein, refer tomaterials separated under conditions that reduce or eliminate thepresence of unrelated materials, i.e., contaminants, including nativematerials from which the material is obtained. For example, a purifiedor isolated protein is preferably Tree of other proteins or nucleicacids with which it can be found within a cell. A purified material maycontain less than about 50%, preferably less than about 75%, and mostpreferably less than about 90%, of the cellular components with which itwas originally associated. Purity can be evaluated by chromatography,get electrophoresis, immunoassay, composition analysis, biological assayand other methods known in the art. From a functional aspect, anisolated GDF8 peptide according to the invention is one sufficientlyseparated from other materials, including precursor GDF8 protein and/ormature GDF8 protein, so as to be capable of eliciting an immune responsethat is specific for the GDF8 peptide.

Methods for purification are well-known in the art. For example, nucleicacids can be purified by precipitation, chromatography,ultracentrifugation and other means. Proteins and polypeptides, as wellas peptides, can be purified by various methods including, withoutlimitation, preparative disc-gel electrophoresis, isoelectric focusing,HPLC, reversed-phase HPLC, gel filtration, ion exchange and partitionchromatography, precipitation and salting-out chromatography, extractionand countercurrent distribution. For some purposes, it is preferable toproduce the polypeptide in a recombinant system in which the proteincontains an additional sequence tag that facilitates purification, suchas, but not limited to, a polyhistidine sequence or a sequence thatspecifically binds to an antibody, such as FLAG® and GST. Thepolypeptide can then be purified from a crude lysate of the host cell bychromatography on an appropriate solid-phase matrix. Alternatively,antibodies, or binding fragments thereof, produced against thepolypeptide can be used as purification reagents.

The term “substantially pure” indicates the highest degree of puritywhich can be achieved using conventional purification techniques knownin the art and means a nucleic acid, polypeptide, peptide, or othermaterial that is free from other contaminating proteins, nucleic acidsand other biologicals derived from an original source organism orrecombinant DNA expression system. Substantial purity may be assayed bystandard methods and wilt typically exceed at least about 75%,preferably at least about 90%, more preferably at least about 95% andmost preferably at least about 99% purity. Purity evaluation may be madeon a mass or molar basis.

A “polynucleotide” or a “nucleic acid molecule” is a molecule comprisingnucleotides including, but is not limited to, RNA, cDNA, genomic DNA andeven synthetic DNA sequences. The terms are also contemplated toencompass nucleic acid molecules that include any of the art-know baseanalogs of DNA and RNA.

A “vector” or “replication vector” is a replicon, such as a plasmid,phage, or cosmid, to which another DNA segment may be attached orincorporated so as to bring about the replication of the attachedsegment. The term also includes a replicon that includes theincorporated or attached DNA segment of interest.

Vectors that can be used in this invention include microbial plasmids,viruses, bacteriophage, integratable DNA fragments and other vehiclesthat may facilitate integration of the nucleic acids into the genome ofthe host. Plasmids are the most commonly used form of vector, but allother forms of vectors which serve an equivalent function and which areor become known in the art are suitable for use herein. See, e.g.,Pouwels et al, Cloning Vectors: A Laboratory Manual 1985 andSupplements, Elsevier, N.Y., and Rodriguez et al. (eds.), Vectors: ASurvey of Molecular Cloning Vectors and Their Uses, 1988, Buttersworth,Boston, Mass.

Insertion of DNA encoding the inventive GDF8 peptide(s) into a vector iseasily accomplished when the termini of both the DNA and the vectorcomprise compatible restriction sites. If this cannot be done, it may benecessary to modify the termini of the DNA and/or vector by digestingback single-stranded DNA overhangs generated by restriction endonucleasecleavage to produce blunt ends, or to achieve the same result by fillingin the single-stranded termini with an appropriate DNA polymerase.Alternatively, desired sites may be produced, e.g., by ligatingnucleotide sequences (linkers) onto the termini. Such linkers maycomprise specific oligonucleotide sequences that define desiredrestriction sites. Restriction sites can also be generated through theuse of the polymerase chain reaction (PCR). See, e.g., Saiki et al.,Science 239:487 (1988). The cleaved vector and the DNA fragments mayalso be modified, if required, by homopolymeric tailing.

Recombinant expression vectors used in this invention are typicallyself-replicating DNA or RNA constructs comprising nucleic acids encodingone of the inventive GDF8 peptide(s) usually operably linked to suitablegenetic control elements that are capable of regulating expression ofthe nucleic acids in compatible host cells. Genetic control elements mayinclude a prokaryotic promoter system or a eukaryotic promoterexpression control system, and typically include a transcriptionalpromoter, an optional operator to control the onset of transcription,transcription enhancers to elevate the level of mRNA expression, asequence that encodes a suitable ribosome binding site, and sequencesthat terminate transcription and translation. Expression vectors mayalso contain an origin of replication that allows the vector toreplicate independently of the host cell.

Expression of nucleic acids encoding inventive GDF8 peptide(s) can becarried out by conventional methods in either prokaryotic or eukaryoticcells.

A DNA “coding sequence” or a “sequence encoding” a particular protein orpeptide, is a DNA sequence which is transcribed and translated into apolypeptide in vitro or in vivo when placed under the control ofappropriate regulatory elements. The boundaries of the coding sequenceare determined by a start codon at the 5′-terminus and a translationstop codon at the 3′-terminus. A coding sequence can include, but is notlimited to, prokaryotic sequences, cDNA from eukaryotic mRNA, genomicDNA sequences from eukaryotic (e.g., mammalian) DNA, and even syntheticDNA sequences. A transcription termination sequence will usually belocated 3′ to the coding sequence.

As used herein the terms “fusion protein” and “fusion peptide” are usedinterchangeably and encompass “chimeric proteins and/or chimericpeptides” and fusion “intein proteins/peptides”. A fusion proteincomprises at least a portion of a GDF8 peptide of the present inventionjoined via a peptide bond to at least a portion of another protein. Forexample, fusion proteins can comprise a marker protein or peptide, or aprotein or peptide that aids in the isolation and/or purification and/orantigenicity of a GDF8 peptide of the present invention. A GDF8 fusionprotein can comprise at least a portion of a non-GDF8 protein joined viaa peptide bond to at least a portion of a GDF8 polypeptide. In preferredembodiments a portion of the GDF8 is functional, i.e, retains itsantigenicity. The non-GDF8 sequences can be amino- or carboxy-terminalto the GDF8 sequences.

A recombinant DNA molecule encoding such a fusion protein comprises asequence encoding at least a portion of a non-GDF8 protein joinedin-frame to the GDF8 coding sequence, and can encode a cleavage site fora specific protease, e.g., thrombin or Factor Xa, preferably at or closeto the juncture between the GDF8 sequence and the non-GDF8 sequence. Ina specific embodiment, the fusion protein is expressed in a CHO cell.Such a fusion protein can be used to isolate the GDF8 peptides of thepresent invention, through the use of an affinity column that isspecific for the protein and/or tag fused to the GDF8 peptide. Thepurified GDF8 peptide for example, may then be released from the fusionprotein through the use of a proteolytic enzyme and a cleavage site suchas has been referred to above.

In one such embodiment, a chimeric GDF8 peptide can be prepared, e.g., aglutathione-S-transferase (GST) fusion protein, a maltose-binding (MBP)protein fusion protein, or a poly-histidine-tagged fusion protein, forexpression in any cell, or alternatively in a cell-free system. Forexample, GST binds glutathione conjugated to a solid support matrix, MBPbinds to a maltose matrix, and poly-histidine chelates to a Ni-chelationsupport matrix. The fusion protein can be eluted from the specificmatrix with appropriate buffers, or by treating with a protease specificfor a cleavage site usually engineered between the GDF8 peptide and thefusion partner (e.g., GST, MBP, FLAG®) as exemplified below, or poly-Hisas described above.

A “heterologous nucleotide sequence”¹ as used herein is a nucleotidesequence that is added to a nucleotide sequence of the present inventionby recombinant methods to form a nucleic acid that is not naturallyformed in nature. Such nucleic acids can encode fusion (e.g., chimeric)proteins. Thus the heterologous nucleotide sequence can encode peptidesand/or proteins that contain regulatory and/or structural properties. Inanother such embodiment the heterologous nucleotide sequence can encodea protein or peptide that functions as a means of detecting the proteinor peptide encoded by the nucleotide sequence of the present inventionafter the recombinant nucleic acid is expressed. In still anotherembodiment the heterologous nucleotide sequence can function as a meansof detecting a nucleotide sequence of the present invention. Aheterologous nucleotide sequence can comprise non-coding sequencesincluding restriction sites, regulatory sites, promoters and the like.

A “host cell” is a cell that contains, or is capable of containing, andexpressing, an exogenous nucleic acid molecule, either transiently orpermanently. A cell has been “transformed” by exogenous DNA when suchexogenous DNA has been introduced inside the cell membrane. ExogenousDNA may or may not be integrated (covalently linked) into chromosomalDNA making up the genome of the cell. In prokaryotes and yeasts, forexample, the exogenous DNA may be maintained on an episomal element,such as a plasmid. With respect to eukaryotic cells, a stablytransformed cell is one in which the exogenous DNA has become integratedinto the chromosome so that it is inherited by daughter cells throughchromosome replication. This stability is demonstrated by the ability ofthe eukaryotic cell to establish cell lines or clones comprised of apopulation of daughter cells containing the exogenous DNA.

Prokaryotes include both gram negative and positive organisms, e.g. E.coli and B. subtilis. Higher eukaryotes include established tissueculture cell lines from animal cells, both of non-mammalian origin, e.g.insect cells, and birds, and mammalian origin, e.g., human, primates,and rodents.

Prokaryotic host-vector systems include a wide variety of vectors formany different species. As used herein, E. coli and its vectors will beused generically to include equivalent vectors used in otherprokaryotes. A representative vector for amplifying DNA is pBR322 ormany of its derivatives. Vectors that can be used to express GDF8,and/or GDF8 peptides, include, but are not limited to, those containingthe lac promoter (pUC-series); trp promoter (pBR322-trp); Ipp promoter(the pIN-series); lambda-pP or pR promoters (pOTS); or hybrid promoterssuch as ptac (pDR540). See Brosius et al., “Expression Vectors EmployingLambda-, trp-, lac-, and lpp-derived Promoters”, in Rodriguez andDenhardt (eds.) Vectors: A Survey of Molecular Cloning Vectors and TheirUses, 1988, Buttersworth, Boston, pp. 205-236.

Yeast, as well as higher eukaryotic tissue culture cells are preferredhosts for the recombinant production of the inventive GDF8 peptides,and/or of anti-GDF8 antibodies and/or fragments of those antibodies.Although any higher eukaryotic tissue culture cell line might be used,including insect baculovirus expression systems, mammalian cells arepreferred. Transformation or transfection and propagation of such cellshave become a routine procedure. Examples of useful cell lines includeHeLa cells, Chinese hamster ovary (CHO) cell lines, baby rat kidney(BRK) cell lines, insect cell lines, bird cell lines, and monkey (COS)cell lines.

Expression vectors for such cell lines usually include, for example, anorigin of replication, a promoter, a translation initiation site, RNAsplice sites (if genomic DNA is used), a polyadenylation site, and atranscription termination site. These vectors also usually contain aselection gene or amplification gene. Suitable expression vectors may beplasmids, viruses, or retroviruses carrying promoters derived, e.g.,from such sources as adenovirus, SV40, parvoviruses, vaccinia virus, orcytomegalovirus. Representative examples of suitable expression vectorsinclude pCR®3.1, pcDNA1, pCD [Okayama et al., Mol. Cell Biol. 5:1136(1985)], pMC1 neo Poly-A [Thomas et al., Cell 51:503 (1987)], pUC19, ppREP8, pSVSPORT and derivatives thereof, and baculovirus vectors, suchas pAC 373 or pAC 610.

Prokaryotic expression control sequences typically used includepromoters, including those derived from the β-lactamase and lactosepromoter Systems [Chang et al., Nature, 198:1056 (1977)], the tryptophan(trp) promoter system [Goeddel et al., Nucleic Acids Res. 8:4057(1980)], the lambda P_(L) promoter system [Shimatake et al., Nature,292:128 (1981)] and the tac promoter [De Boer et al., Proc, Natl. Acad.Sci, USA 292:128 (1983)]. Numerous expression vectors containing suchcontrol sequences are known in the art and commercially available.

“Operably linked” refers to an arrangement of elements wherein thecomponents so described are configured so as to perform their usualfunction. Thus, control elements operably linked to a coding sequenceare capable of effecting the expression of the coding sequence. Thecontrol elements need not be contiguous with the coding sequence, solong as they function to direct the expression thereof. Thus, forexample, intervening untranslated yet transcribed sequences can bepresent between a promoter and the coding sequence and the promoter canstill be considered “operably linked” to the coding sequence.

The invention also includes polyclonal and monoclonal (mAb) antibodiesthat specifically bind to the inventive GDF8 peptides. As used herein,the term “antibody” refers to an immunoglobulin and/or fragmentsthereof. A naturally occurring immunoglobulin consists of one or morepolypeptides substantially encoded by immunoglobulin genes. Therecognized immunoglobulin genes include the kappa, lambda, alpha, gamma,delta, epsilon and mu constant region genes, as well as the myriadimmunoglobulin variable region genes. An antibody or antibodiesaccording to the invention also encompass antibody fragments, i.e.,antigen-binding fragments, for example, Fv, Fab, and F(ab′)₂, engineeredsingle-chain binding proteins, (e.g., Huston et al., Proc. Natl. Acad.Sci. U.S.A., 85, 5879-5883 (1988) and Bird et al., Science, 242, 423-426(1988), hereby incorporated herein by reference in its entireties), aswell as bifunctional hybrid antibodies (e.g., Lanzavecchia et al., Eur.J. Immunol. 17, 105 (1987)). [See, generally, Hood et al., Immunology.Benjamin, N.Y., 2nd ed. (1984), Harlow and Lane, Antibodies. ALaboratory Manual, Cod Spring Harbor Laboratory (1988) and Hunkapillerand Hood, Nature, 323, 15-16 (1986), the contents of all of which arehereby incorporated by reference in their entireties.]

For example, serum produced from animals immunized by the inventive GDF8peptides, using standard methods, can be used directly, or the IgGfraction can be separated from the serum using standard methods, such asplasmaphoresis or adsorption chromatography with IgG-specificadsorbents, such as immobilized Protein A or Protein G. Alternatively,monoclonal antibodies can be prepared, and optionally antigen bindingfragments or recombinant binding proteins derived from such mAbs. SuchMAbs or fragments thereof can optionally be humanized by art-knownmethods.

Hybridomas producing mAbs that selectively bind the GDF8 peptides of theinvention, are produced by well-known techniques. Usually, the processinvolves the fusion of an immortalizing cell line with a B-lymphocytethat produces the desired antibody. Alternatively, non-fusion techniquesfor generating immortal antibody-producing cell lines can be used, e.g.,virally-induced transformation [Casali et al., Science 234:476 (1986)].Immortalizing cell lines are usually transformed mammalian cells,particularly myeloma cells of rodent, bovine, and human origin. Mostfrequently, rat or mouse myeloma cell lines are employed as a matter ofconvenience and availability.

Techniques for obtaining antibody-producing lymphocytes from mammalsinjected with antigens are well known. Generally, peripheral bloodlymphocytes (PBLs) are used if cells of human origin are employed, orspleen or lymph node cells are used from non-human mammalian sources. Ahost animal is injected with repeated dosages of the purified antigen(human cells are sensitized in vitro), and the animal is permitted togenerate the desired antibody-producing cells before they are harvestedfor fusion with the immortalizing cell line. Techniques for fusion arealso well known in the art, and, in general, involve mixing the cellswith a fusing agent, such as polyethylene glycol.

Hybridomas are selected by standard procedures, such as HAT(hypoxanthine-aminopterin-thymidine) selection. Those secreting thedesired antibody are selected using standard immunoassays, such asWestern blotting, ELISA (enzyme-linked immunosorbent assay), RIA(radioimmunoassay) or the like. Antibodies are recovered from the mediumusing standard protein purification techniques [Tijssen, Practice andTheory of Enzyme Immunoassays (Elsevier, Amsterdam, 1985)].

Many references are available to provide guidance in applying the abovetechniques [Kohler et al. Hybridoma Techniques (Cold Spring HarborLaboratory, New York, 1980); Tijssen, Practice and Theory of EnzymeImmunoassays (Elsevier, Amsterdam, 1985); Campbell, Monoclonal AntibodyTechnology (Elsevier, Amsterdam, 1984); Hurrell, Monoclonal HybridomaAntibodies: Techniques and Applications (CRC Press, Boca Raton, Fla.,1982]. Monoclonal antibodies can also be produced using well-known phagelibrary systems. See, e.g., Huse, et al., Science 246:1275 (1989); Ward,et al., Nature, 341:544 (1989).

Antibodies thus produced, whether polyclonal or monoclonal, can be used,e.g., in an immobilized form bound to a solid support by well knownmethods to purify the GDF8 peptides by immunoaffinity chromatography.

Antibodies against the GDF8 peptides can also be used, unlabeled orlabeled by standard methods, as the basis for immunoassays to detect orquantify GDF8. The particular label used will depend upon the type ofimmunoassay. Examples of labels that can be used include, but are notlimited to, radiolabels, such as ³²P, ¹²⁵I, ³H and ¹⁴C; fluorescentlabels, such as fluorescein and its derivatives, rhodamine and itsderivatives, dansyl and umbelliferone; chemiluminescers, such asluciferia and 2,3-dihydrophthalazinediones; and enzymes, such ashorseradish peroxidase, alkaline phosphatase lysozyme andglucose-6-phosphate dehydrogenase.

The antibodies can be tagged with such labels by known methods. Forexample, coupling agents such as aldehydes, carbodiimides, dimaleimide,imidates, succinimides, bisdiazotized benzadine and the like may be usedto tag the antibodies with fluorescent, chemiluminescent or enzymelabels. The general methods involved are well known in the art and aredescribed, e.g., in Immunoassay: A Practical Guide, 1987, Chan (Ed.),Academic Press, Inc., Orlando, Fla. Such immunoassays could be carriedout, for example, on fractions obtained during purification of thereceptors.

The antibodies of the present invention can also be used to identifyparticular cDNA clones expressing GDF8-related polypeptides inexpression cloning systems. Neutralizing antibodies specific for theligand-binding site of a receptor can also be used as antagonists(inhibitors) to block or downregulate GDF8 function. Such neutralizingantibodies can readily be identified through routine experimentation, asexemplified by the Examples provided below.

Antagonism of GDF8 activity can be accomplished using complete antibodymolecules, or well-known antigen binding fragments such as Fab, Fc,F(abL)₂ and Fv fragments, Definitions of such fragments can be found asdescribed hereinabove, or e.g. in Klein, Immunology (John Wiley, NewYork, 1982); Parham, Chapter 14, in Weir, ed. Immunochemistry, 4th Ed,(Blackwell Scientific Publishers, Oxford, 1986). The use and generationof antibody fragments has also been described, e.g.: Fab fragments[Tijssen, Practice and Theory of Enzyme Immunoassays (Elsevier.Amsterdam, 1985)], Fv fragments [Hochman et al., Biochemistry 12:1130(1973); Sharon et al., Biochemistry 15:1591 (1976): Ehrlich et al., U.S.Pat. No. 4,355,023] and antibody half molecules (Auditore-Hargreaves.U.S. Pat. No., 4,470,925). Methods for making recombinant Fv fragmentsbased on known antibody heavy and light chain variable region sequenceshave further been described. e.g. by Moore et al. (U.S. Pat. No.4,642,334) and by Pluckthun [Bio/Technology 9:545 (1991)].Alternatively, they can be chemically synthesized by standard methods.

The present invention also encompasses anti-idiotypic antibodies, bothpolyclonal and monoclonal, which are produced using the above-describedantibodies as antigens. These antibodies are useful because they maymimic the structures of the ligands.

Peptide Synthesis

Since the inventive GDF8 peptides, e.g., the DJ5 peptide exemplifiedhereinbelow, are relatively short (e.g., preferably 50 amino acidresidues or less), they may be prepared by art-known methods of peptidesynthesis. Synthetic peptides or polypeptides, prepared using thewell-known techniques of solid phase, liquid phase, or peptidecondensation techniques, or any combination thereof, can include naturaland unnatural amino acids, Amino acids used for peptide synthesis may bestandard Boc (N_(alpha)-amino protected N_(alpha)-t-butyloxycarbonyl)amino acid resin with the standard deprotecting, neutralization,coupling and wash protocols of the original solid phase procedure ofMerrifield [J. Am. Chem. Soc., 85:2149-2154 (1963)], or the base-labileN_(alpha)-amino protected 9-fluorenylmethoxycarbonyl (Fmoc) amino acidsfirst described by Carpino and Han [J. Org. Chem., 37:3403-3409 (1972)].Both Fmoc and Boc N_(alpha)-amino protected amino acids can be obtainedfrom Fluka, Bachem, Advanced Chemtech, Sigma, Cambridge ResearchBiochemical, Bachem, or Peninsula Labs or other chemical companiesfamiliar to those who practice this art. In addition, the method of theinvention can be used with other N_(alpha)-protecting groups that arefamiliar to those skilled in this art. Solid phase peptide synthesis maybe accomplished by techniques familiar to those in the art and provided,for example, in Stewart and Young, SOLID PHASE SYNTHESIS, SecondEdition, Pierce Chemical Co., Rockford, Ill., (1984); Fields and Noble,Int. J. Pept. Protein Res. 35:161-214 (1990), or using automatedsynthesizers, such as sold by ABS [Applied Biosystems, 850 LincolnCentre Drive, Foster City, Calif. 94404 USA]. Thus, the GDF8 peptides ofthe invention may comprise D-amino acids, a combination of D- andL-amino acids, and various “designer” amino acids (e.g., beta-methylamino acids, C_(alpha)-methyl amino acids, and N_(alpha)-methyl aminoacids, etc., to convey special properties. Synthetic amino acid includeornithine for lysine fluorophenylalanine for phenylalanine, andnorleucine for leucine or isoleucine. Additionally, by assigningspecific amino acids at specific coupling steps, alpha-helices, betaturns, beta sheets, gamma-turns, and cyclic peptides can be generated.

Anti-GDF8 Antiserum

The methods of the invention included a process of screening GDF8peptides against a polygonal anti-GDF8 antiserum. This processidentified the epitope that certain anti-GDF8 antibodies bind to in ahighly specific way. The anti-GDF8 antiserum was obtained by immunizingan animal with precursor GDF8. The precursor GDF8 gene was modified toprovide a form optimized for expression and immunogenicity. For example,the natural DNA sequence of the GDF8 prohormone (SEQ ID NO: 2) wasoptimized for expression in mammalian and viral expression systems. Inaddition, changes were made to avoid the negative effects of viral hostshutoff mechanisms. Typically viral host shutoff mechanisms involvetranscriptional control, RNA stability (splicing) and such. Thesechanges made the nucleic acid less host like and more virus like.

Further, the DNA sequence was preferably designed to be as divergentfrom the mammalian nucleic acid sequence as possible. For example, theamino acid sequence of the precursor GDF8 was reverse translated usingyeast preferred codons. The resulting sequence was surveyed for codonsthat retained their homology to the human GDF8 nucleic acid sequence.Where possible these codons were substituted with the next mostpreferred yeast codons encoding the same amino acid.

The resulting optimized gene (SEQ ID NO: 3) can be expressed in anysuitable host system, including, e.g., art-known insect, mammalian,bacterial, viral and yeast expression systems. For example, insect cellexpression systems, such as baculovirus systems, are art-known anddescribed, for instance, by Summers and Smith, Texas AgriculturalExperiment Station Bulletin No. 1555 (1987). Materials and methods forbaculovirus/insect cell expression systems are commercially available inkit form from, inter alia, Invitrogen, San Diego Calif. (“MaxBac” kit).Similarly, bacterial and mammalian cell expression systems are wellknown in the art and described, for example, by Sambrook et al.(MOLECULAR CLONING: A LABORATORY MANUAL; DNA Cloning, Vols. I and II: D.N. Glover ed.). Yeast expression systems are also known in the art anddescribed, for example, by, YEAST GENETIC ENGINEERING (Barr et al.,eds., 1989) Butterworths, London, Many other such expression systems areknown to the art and are available commercially in kit form. Asexemplified herein, the modified precursor GDF8 gene (SEQ ID NO: 3) wasexpressed in a Flp-In™ CHO expression system (invitrogen, Carlsbad,Calif.) as described in greater detail by Example 1 below.

The precursor GDF8 protein, as well as peptides of the mature GDF8protein, can be incorporated into any protein- or peptide-compatiblevaccine composition. Such vaccine compositions are well known to the artand include, for example, physiologically compatible buffers and salineand the like, as well as adjuvants, as described in greater detailhereinbelow. The vaccine composition including the precursor GDF8protein is employed for eliciting antiserum for screening andidentifying a specific neutralization epitope for an anti-GDF8 antibody.

As exemplified herein, purified precursor GDF8 protein expressed by avector comprising SEQ ID NO:3 was injected into a goat in a vaccinecomposition that included 1 g of precursor GDF8 protein emulsified intoFreund's complete adjuvant (CFA). The vaccine composition was preferablyinjected subcutaneously (SC) beneath the skin of the goat. Subsequentbooster immunizations are preferred. These can be administered atsuitable additional intervals with the same or a reduced dosage of theprotein, e.g., at intervals ranging from 2-5 weeks after the initialinjection.

Beginning from about two weeks after the initial injection, butpreferably starting after a longer time period, e.g., from three tofifteen weeks, or longer, serum is collected, as needed, from theimmunized animal. The collected serum is then preferably purified and/orfractionated by conventional immunoglobulin purification procedures suchas, for example, protein A-Sepharose, protein O-agarose, hydroxylapatitechromatography, gel electrophoresis, dialysis, or affinitychromatography with a suitable ligand. As exemplified herein, the IgGfraction of the serum was further fractionated on a protein G-agarosecolumn.

The anti-GDF8 antiserum IgG fraction is then available for screeningagainst a range of peptides of the mature GDF8, as described in greaterdetail by Example 3, hereinbelow.

GDF8 Binding Epitopes

Suitable anti-GDF8 monoclonal or polyclonal antibodies were contactedwith GDF8 protein for a time period sufficient for the antibody to bindselectively to the protein. Thereafter, ODES bioassays conformed thatthe antibody neutralized substantially all of the GDF8 protein activity.Any GDF8 bioassay can be employed for this purpose, although, asexemplified hereinbelow by Example 3, an in vitro transcriptionalactivation assay according to Thies et. al., 2001, (Growth Factors 18,251) is preferred.

Generally, a GDF8 peptide useful as an antigen or binding epitopeaccording to the invention includes from about residue 312 to aboutresidue 361 of GDF8 (SEQ ID NO: 1). In particular, a peptide accordingto the invention includes from about residue 320 to about residue 350 ofGDF8 (SEQ ID NO: 1). The peptide preferably includes from about residue327 to about residue 346 of GDF8 (SEQ ID NO: 1).

The artisan will appreciate that the inventive GDF8 peptide can bereadily modified to include at least one conservative amino acidsubstitution, and at any position. Preferably, that peptide specificallybinds to rat monoclonal antibody 788, as exemplified hereinbelow. Suchconservative substitutions can include, for example, variations atresidues 328, 329 and 335, and combinations thereof, wherein, amino acidresidue 328 is His, Leu, Asn or Val, amino acid residue 329 is Lys orLeu; and amino acid residue 335 is Ser or Pro or Thr. Precursor GDF8residues 328, 329 and 335 vary within the GDF8 protein sequence acrossspecies, as illustrated by FIG. 2, but nevertheless, the mature GDF8remains functional.

FIG. 1 illustrates a map of the GDF8 active region (that forms themature protein) in the context of its precursor protein. Superimposed onthe map of the GDF8 active region are the locations of seven overlappingpeptides. These overlapping peptide were designed in order to providetargets for identifying the antibody-binding epitope or epitopes ofGDF8. The peptide labeled as DJ5 was identified by screening with theIgG fraction of the exemplified goat anti-GDF8 antiserum as the onlysignificant binding epitope of GDF8 for the exemplified antiserum. Thispeptide has a sequence (SEQ ID NO: 8) corresponding to residue 327 toresidue 346 of precursor GDF8 (SEQ ID NO: 1).

GDF8 Peptide Vaccine Compositions

The GDF8 peptides described above are preferably formulated into vaccinecompositions. These vaccine compositions may be employed to immunize ananimal in order to elicit a highly specific anti-GDF8 immune response.The result of the immunization will be downregulation of GDF8 functionin the immunized animal. Such vaccine compositions are well known to theart and include, for example, physiologically compatible buffers,preservatives, and saline and the like, as well as adjuvants.

“Adjuvants” are agents that nonspecifically increase an immune responseto a particular antigen, thus reducing the quantity of antigen necessaryin any given vaccine, and/or the frequency of injection necessary inorder to generate an adequate immune response to the antigen ofinterest. Suitable adjuvants for the vaccination of animals include, butare not limited to, Adjuvant 65 (containing peanut oil, mannidemonooleate and aluminum monostearate); Freund's complete or incompleteadjuvant; mineral gels, such as aluminum hydroxide, aluminum phosphateand alum; surfactants, such as hexadecylamine, octadecylamine,lysolecithin, dimethyldioctadecylammonium bromide,N,N-dioctadecyl-N′,N′-bis(2-hydroxymethyl) propanediamine,methoxyhexadecylglycerol and pluronic polyols; polyanions, such aspyran, dextran sulfate, poly IC, polyacrylic acid and carbopol;peptides, such as muramyl dipeptide, dimethylglycine and tuftsin; andoil emulsions. The protein or peptides could also be administeredfollowing incorporation into liposomes or other microcarriers.Information concerning adjuvants and various aspects of immunoassays aredisclosed, e.g., in the series by P. Tijssen, Practice and Theory ofEnzyme Immunoassays, 3rd Edition, 1987, Elsevier, N.Y., incorporated byreference herein.

The vaccine composition includes a sufficient amount of the desiredimmunogen, such as the inventive GDF8 peptides, to elicit an immuneresponse. The amount administered can range from about 0.0001 g/kg toabout 1.0 g/kg, relative to the mass of the animal. Any suitablevertebrate animal is readily employed to obtain polyclonal antiserum.Preferably, the animal is a mammal, and includes, but is not limited to,rodents, such as a mice, rats, rabbits, horses, canines, felines,bovines, ovines, e.g., goats and sheep, primates, e.g., monkeys, greatapes and humans, and the like.

The vaccine composition is readily administered by any standard route,including intravenously, intramuscularly, subcutaneously,intraperitoneally, in ovo (particularly for poultry), and/or orally, Forfish species, methods of administering a vaccine composition orimmunogenic composition include the foregoing, as well as dipping thefish into water comprising an antigenic concentration of the peptide,spraying the fish with in antigenic concentration of the peptide whilethe fish is briefly separated from the water, etc. The artisan willappreciate that the vaccine composition is preferably formulatedappropriately for each type of recipient animal and route ofadministration.

Appropriate animal subjects can include those in the wild, livestock(e.g., raised for meat, milk, butter, eggs, fur, leather, feathersand/or wool), beasts of burden, research animals, companion animals, aswell as those raised for/in zoos, wild habitats and/or circuses. In aparticular embodiment, the animal is a great ape such as a gorilla, or ahuman.

In one preferred embodiment, the animal is a “food-producing” animal,and the result of immunization is a gain in animal weight, particularlymuscle mass, relative to animals not immunized. For purposes of thepresent invention, the term “food-producing” animal shall be understoodto include all animals bred for consumption by humans and/or otheranimals, or for producing consumables such as eggs or milk. Anon-limiting list of such animals include avians (e.g., chickens,turkeys, ducks, geese, ostriches), bovine (e.g., beef/veal cattle, dairycows, breeding bulls, buffalo), porcine (e.g., hogs or pigs), ovine(e.g., goats or sheep), equine (e.g., horses) as well as aquaticanimals, including shellfish and fish such as trout or salmon, and otherspecies raised or harvested for human consumption.

For purposes of the present invention, the term “fish” shall beunderstood to include without limitation, the Teleosti grouping of fish,i.e., teleosts. Both the Salmoniformes order (which includes theSalmonidae family) and the Perciformes order (which includes theCentrarchidae family) are contained within the Teleosti grouping.

Examples of potential fish recipients include the Salmonidae family, theSerranidae family, the Sparidae family, the Cichlidae family, theCentrarchidae family, the three-Line Grunt (Parapristipoma trilineatum),and the Blue-Eyed Plecostomus (Plecostomus spp).

Salmonidae Family TAXON NAME COMMON NAME Coregonus clupeaformis Lakewhitefish Coregonus hoyi Bloater Oncorhynchus keta Chum salmonOncorhynchus gorbuscha Pink salmon Oncorhynchus kisutch Coho salmon(silver salmon) Oncorhynchus masou cherry salmon (masou salmon)Oncorhynchus nerka Sockeye salmon Oncorhynchus tshawytscha (chinooksalmon) Prosopium cylindraceum Round whitefish Oncorhynchus clarkiCutthroat trout Oncorhynchus mykiss Rainbow trout Salmo salar Atlanticsalmon Salmo trutta Brown trout Salmo trutta X S. fontinalis Tigerhybrid-trout Salvelinus alpinus Arctic charr Salvelinus confluentus Bulltrout Salvelinus fontinalis Brook trout Salvelinus leucomaenis Japanesecharr (white spotted charr) Salvelinus malma Dolly varden (Miyabe charr)Salvelinus namaycush Lake trout Thymallus thymallus Grayling

Some Members of the Serranidae Family TAXON NAME COMMON NAMECentropristis ocyurus Bank sea bass Centropristis philadelphicus Rocksea bass Centropristis striata Black sea bass Diplectrum bivittatumDwarf sandperch Diplectrum formosum Sand perch Epinephelus flavolimbatusYallowedge grouper Epinephelus morio Red grouper Serranus phoebe TattlerSerranus tortugarum Chalk bass

Some Members of the Sparidae family TAXON NAME COMMON NAME Archosargusprobatocephalus Sheepshead Archosargus rhomboidalis Sea bream Calamuspenna Sheepshead porgy Lagodon rhomboides Pinfish Pagrus Major Red Seabream Sparus aurata Gilthead Sea bream Stenotomus chrysops Scup

Some Members of the Cichlidae family TAXON NAME COMMON NAME Aequidenslatifrons Blue acara Cichlisoma nigrofasciatum Congo cichlidCrenichichla sp. Pike cichlid Pterophyllum scalare Angel fish Tilapiamossambica Mozambique mouth breeder Oreochromis sp. Tilapia Sarotherodonaurea Golden Tilapia

Some Members of the Centrarchidae family TAXON NAME COMMON NAMEAmbloplites rupestris Rock bass Centrarchus macropterus Flier Elassomaevergladei Everglades pigmy sunfish Elassoma okefenokee Okefenokee pigmysunfish Elassoma zonatum Banded pigmy sunfish Enneacanthus gloriosusBluespotted sunfish Enneacanthus obesus Banded sunfish Lepomis auritusRedbreast sunfish Lepomis cyanellus Green sunfish Lepomis cyanellus X L.gibbosus Green × pumpkinseed Lepomis gibbosus Pumpkinseed Lepomisgulosus Warmouth Lepomis humilis Orange-spotted sunfish Lepomismacrochirus Bluegill Lepomis megalotis Longear sunfish Micropteruscoosae Shoal bass Micropterus dolomieui Smallmouth bass Micropteruspunctulatus Spotted bass Micropterus salmoides Largemouth bass Pomoxisannularis White crappie Pomoxis nigromaculatus Black crappie

In a further preferred embodiment, the animal is a companion animal or ahuman, and the vaccine is administered to provide long-termdownregulation of GDF8 for any veterinary or medical purpose responsiveto such GDF8 downregulation. For purposes of the present invention, theterm “companion” animal shall be understood to include allanimals—horses (equine) cats (feline), dogs (canine) rodents, (includingmice, rats, guinea pigs), rabbit species, and avians, such as pigeons,parrots and the like.

Other birds receiving such vaccination or antibodies car be associatedwith either commercial or noncommercial aviculture. These include e.g.,Anatidae, such as swans, Columbidae, e.g., doves and pigeons, such asdomestic pigeons, Phasianidae, e.g., partridge, and grouse, Thesienidae,Psittacines, e.g., parakeets, macaws, and parrots, e.g., raised for thepet or collector market, and members of the Ratite family.

In another preferred embodiment, any of the above recited animals(preferably nonhuman) are immunized in order to obtain anti-GDF8antibodies that specifically bind to the inventive peptides, and theelicited antibodies are harvested for use in assays, and/or inveterinary or human medicine, e.g., to provide downregulation of GDF8for any veterinary or medical purpose responsive to such GDF8downregulation.

The present invention may be better understood by reference to thefollowing non-limiting examples, which are provided as exemplary of theinvention. The following examples are presented in order to more fullyillustrate embodiments of the invention and should in no way beconstrued as limiting the broad scope of the invention.

EXAMPLES Example 1 Materials & Methods A. Expression and Purification ofPrecursor GDF8 (GDF8 Prohormone)

The natural DNA sequence of the precursor GDF8 or prohormone (SEQ ID NO:2) was optimized for expression in mammalian and viral expressionsystems. To avoid the negative effects of viral host shutoff mechanismsthe DNA sequence was designed to be as divergent from the mammaliannucleic acid sequence as possible. To accomplish this the amino acidsequence of the GDF8 prohormone was reversed translated using yeastpreferred codons. The resulting sequence was surveyed for codons, whichretained their homology to the human GDF8 nucleic acid sequence. Wherepossible these codons were substituted with the next most preferredyeast codons encoding the same amino acid. The resulting nucleic acidmolecule (SEQ ID NO: 3) was commercially synthesized for incorporationinto the appropriate expression vectors.

The Flp-In™ CHO expression system (Invitrogen. Carlsbad, Calif.) wasused to express the optimized GDF8 prohormone. Briefly, a GDF8prohormone construct containing a C-terminal FLAG® (Sigma-Aldrich Corp,St Louis. MO) epitope fusion was constructed by inserting the geneencoding the modified GDF8 prohormone into plasmid pCMVtag4B(Stratagene, San Diego. CA.). The FLAG® fusion tag facilitatesseparation of FLAG® fusion proteins on an anti-FLAG® gel column. A PCRDNA fragment containing the modified GDF8 prohormone-FLAG® gene was thencloned into plasmid expression vector pcDNA5/FRT (Invitrogen, Carlsbad,Calif.). Generation of the Flp-In™ CHO cell line expressing the GDF8prohormone-FLAG® fusion protein was achieved by cotransfection of theFlp-In™ CHO cell line with the Flp-In™ expression vector containing theGDF8-FLAG® gene and the Flp recombinase expression plasmid, POG44. Ftprecombinase mediates insertion of the Flp-In expression cassette intothe genome at an integrated FRT site by site-specific DNA recombination.A stable cell line expressing and secreting the GDF8 prohormonecontaining the FLAG® epitope was obtained using hygromycin B selection.

The stable CHO cell line expressing the GDF8 prohormone containing theFLAG® tag was adapted to suspension culture in serum-free media usingstandard techniques. Conditioned media containing the secreted GDF8prohormone was generated using the WAVE bioreactor system (WAVE BiotechLLC, Bridgewater, N.J.). Purification of the FLAG® tagged GDF8prohormone was achieved by affinity chromatography using an anti-FLAG®M2 affinity gel (Sigma-Aldrich Corp., St. Louis. MO).

B. DJ5 Specific Antibody Purification

DJ5 (SEQ ID NO: 8; See Table 2, below) specific antibody fractions werepurified by affinity column chromatography. An affinity column wasprepared by coupling 10 mg of DJ5 synthetic peptide to 0.8 g of cyanogenbromide-activated Sepharose 4B (Sigma Genosys, Woodlands, Tex.), Thecolumn was washed and equilibrated with PBS. Approximately 11 mm of GoatIgG fraction (10 mg/ml) was applied to the affinity column and washedwith 25 ml of PBS. Fractions of 1.0 ml were collected and monitored forabsorbance at 280 nm. Bound material was eluted with approximately 10 mlof 0.2 M glycine (pH 1.85). Fractions of 1.0 ml were collected andneutralized with 0.25 ml of 0.5 M sodium phosphate, 0.75 M NaCl, pH 7.4.Approximately 25 μl aliquots of unbound fractions 1-10 and boundfractions 25-35 were assayed for ELISA reactivity to DJ5 peptide.Unbound fractions were found to be negative for DJ5 reactivity Boundfractions exhibited a strong peak of reactivity to the DJ5 peptide.Unbound fractions 1-11 and bound fractions 26-34 were pooled. Pooledsamples were concentrated and their buffer exchanged with phosphatebuffered saline (PBS) as indicated below. Sample concentrations weredetermined by the OD 280 method (CURRENT PROTOCOLS IN IMMUNOLOGY, 2.7.3,John Wiley & Sons, Inc.). The unbound sample was adjusted to 10 mg/mland the bound sample was adjusted to 1 mg/ml, for subsequent use.

Example 2 Goat Anti-GDF8 Polyclonal IgG Serum

Goat anti-precursor GDF8 IgG was obtained from an immunized goat by thefollowing methods.

A. Immunization of Goat

A Saanen (dairy) goat (approximately 2 year old male) was immunized withpurified recombinant GDF8 prohormone (obtained as described by Example1, above) as follows. One half mg of protein was emulsified in Freund'scomplete adjuvant (CFA) and injected subcutaneously (SC) beneath theskin of the goat. Subsequent booster immunizations administered SC atweeks three, six, and ten contained 0.3 mg of protein emulsified inFreund's incomplete adjuvant (IFA). Blood was collected from the jugularvein with a syringe and needle, and taken with vacuum bottle and tubing.The blood was collected in bottles containing anticoagulant andcentrifuged at 2500 RPM for 20 minutes to remove the red blood cells.The plasma was re-calcified to produce serum. The serum sample collected15 weeks post initial immunization was used for further analysis.

B. Collection and Purification of Goat Polyclonal IgG

Serum was harvested from the goat after 15 weeks, and the IgG fractionwas purified from this serum, as follows. The IgG fraction of goat serawas purified on a Protein G agarose column according to the manufacturesprotocol (Kirkegaard and Perry Laboratories, Inc., Gaithersburg, Md.).Eluted fractions were pooled, concentrated, and buffer exchanged withphosphate buffered saline (PBS) utilizing Centriprep centrifugal Filters(Centriprep YM-10, Miilipore Corporation, Billerica, Mass.). Sampleconcentrations were determined by the OD 280 method (CURRENT PROTOCOLSAN IMMUNOLOGY, Id.) and adjusted to 10 mg/ml.

Example 3 Characterization of Goat Antiserum

The goat antiserum provided by Example 2, above, is designated as PGA.It is expected that the PGA IgG fraction contains antibodies directedagainst various epitopes on the GDF8 prohormone molecule. The PGAantiserum was characterized by an in vitro transcription activationassay, as follows. The in vitro transcriptional activation assay used toquantitatively measure GDF8 bio-neutralization is essentially that ofThies et. al. (Growth Factors 18, 251 (2001)), Ninety-six well tissueculture treated luminometer ViewPlate™ assay plates (PerkinElmer Lifeand Analytical Sciences, Inc., Boston, Mass.) were seeded with 1.0×10⁵cells/well of A204 Rhabdomyosarcoma cells (ATCC HTB-82) and incubated ina 37° C., 5% CO2, humidified chamber. Complete A204 culture mediaconsists of McCoy's 5A medium, 10% fetal bovine serum, 2% L-glutamine,and 1% Penn/Strep. Upon reaching greater than 80% confluence, the cellswere transiently transfected with a mixture of plasmid pDPC4-luciferaseand HCMV IE-lacZ using the protocol recommended by the manufacturer ofthe FUGENE transfection reagent (Roche Diagnostics Corporation,Indianapolis, Ind.) and incubated 16 hours in a 37° C., 5% CO2,humidified chamber. Plasmid pDPC4-luciferase contains four copies of theCAGA box, derived from the human plasminogen activator inhibitor(PAI-1), which confers GDF8 responsiveness to the heterologous promoterreporter construct.

Plasmid HCMV IE-lacZ contains a beta-galactosidase gene under thecontrol of the constitutive human cytomegalovirus immediate earlypromoter. This gene is added as a control to normalize for transfectionefficiencies. Cells were then treated with 100 ng/well GDF8 protein (R&DSystems Inc., Minneapolis, Minn.) and incubated an additional 16 hoursin a 37° C. 5% CO2, humidified chamber. Luciferase andbeta-galactosidase were quantified in the treated cells using theDual-Light Luciferase Assay (Tropix, Applied Biosystems, Foster City,Calif.).

Each sample was run in duplicate (2 wells). The signal for each well wascalculated as the luciferase signal divided by the beta-glactosidasesignal times 100. The sample signal was calculated as the average of thetwo wells.

To test the bio-neutralization activity of an antibody sample variousconcentrations of purified IgG fractions were incubated with the GDF8protein (approximately 16 hours at 4° C.) prior to treatment of thecells. The percent inhibition was calculated as 100−(100× samplesignal)/(signal with GDF8 alone—signal with no GDF8 added). The resultsof the in vitro transcription activation assay are summarized by Table1, below.

TABLE 1 GDF8 neutralization titers for Goat Serum PGA Sample (μg IgG) %Inhibition of GDF8 Activity Goat - normal (250) 0 Goat - PGA (250) 95Goat - PGA (125) 86 Goat - PGA (63) 62 Goat - PGA (31) 22 Goat - PGA(16) 3

The neutralization assay confirmed that the IgG fraction of theharvested goat serum contains antibodies capable of neutralizing atleast 95% of the GDF8 used in this activity assay.

Example 4 Goat Polyclonal Antibody Defines A Specific NeutralizationEpitope of the GDF8 Protein

In order to determine the specificity of the neutralizing immuneresponse the PGA IgG fraction was assayed for its reactivity with a setof seven overlapping peptides (DJ1-7 see Table 2 and FIG. 1) that spanthe entire coding region of the active GDF8 protein. Reactivity of theGoat PGA IgG to each individual peptide was determined by Enzyme-Linkedimmunosorbant Assay (ELISA) assay. The GDF8 peptide ELISA was performedessentially as described in Protein Detector™ ELISA Kit HRP, ABTS System(Kirkegaard and Perry Laboratories, Inc., Gaithersburg, Md.). Thefollowing modifications were used in the assay. Synthetic peptides DJ1-7(see Table 2, below) were custom synthesized under our direction byProSci, Inc. (Poway, Calif.). Plates were coated with synthetic peptidesat 500 ng per well and purified GDF8 prohormone at 250 ng per well.Primary antibodies were IgG fractions from various samples. Secondaryantibodies were used at a dilution of 1:2000. For goat primary antibodysamples the secondary antibody was rabbit peroxidase-labeled antibody togoat IgG. For rat primary antibody samples the secondary antibody wasgoat peroxidase-labeled antibody to rat IgG. The OD 405 nm was read or15 minutes with an ELISA plate reader. The ELISA reactivity wascalculated as OD 405 per minute times 1000.

TABLE 2 GDFS Active Region Peptides Coordi- Name nates* Amino acidsequence DJ1 267-286 DFGLDCDEHSTESRCCRYPL SEQ ID NO: 4 DJ2 282-301CRYPLTVDFEAFGWDWIIAP SEQ ID NO: 5 DJ3 297-316 WIIAPKRYKANYCSGECEFV SEQID NO: 6 DJ4 312-331 ECEFVFLQKYPHTHLVHQAN SEQ ID NO: 7 DJ5 327-346VHQANPRGSAGPCCTPTKMS SEQ ID NO: 8 DJ6 342-361 PTKMSPINMLYFNGKEQIIY SEQID NO: 9 DJ7 357-375 EQIIYGKIPAMVVDRCGCS SEQ ID NO: 10 *relative toHuman GDF8 prohormone (Genebank Accession Number NP_005250)

The ELISA results are summarized by Table 3, below.

TABLE 3 ELISA reactivity of PGA IgG (10 mg/ml) to GDF8 Active RegionPeptides OD 405/ minute × 1000 Antigen 1:20 1:40 1:80 DJ1* 23 10 1 DJ2*3 0 0 DJ3* 0 10 0 DJ4* 3 0 0 DJ5* 121 37 27 DJ6* 3 0 0 DJ7* 10 1 0proGDF8** 194 196 199 *peptide, **prohormone

The PGA IgG fraction reacted specifically with both the purified GDF8prohormone and with the DJ5 peptide. Among the GDF8 active regionpeptides the IgG fraction reacts specifically and exclusively with theDJ5 peptide. This is a strong indication that the neutralizingcapability of this serum is directed against an epitope defined by theDJ5 peptide. In order to confirm this hypothesis the DJ5 specificfraction of PGA IgG was purified. This was accomplished by affinitychromatography as described in the materials and methods. The PGAantibodies were separated into DJ5 peptide bound and unbound fractions.Both fractions were assayed for neutralization activity against the GDF8protein.

The results in Table 4 show that the majority of GDF8 neutralizationcapacity resides with the antibody that binds specifically to the DJ5peptide. This clearly demonstrates that the DJ5 peptide defines aneutralizing epitope of the GDF8 protein.

TABLE 4 GDF8 neutralization activity of DJ5 specific antibodies Sample(μg IgG) % Inhibition of GDF8 Activity Goat-normal (250)** 7 DJ5 unboundIgG (250) 26 DJ5 bound IgG (25) 90 **The normal goat IgG was a negativecontrol purified from non-immunized goat sera (commercially purchased).

Curiously, in a preliminary experiment, neutralizing GDF8 antibodieswere not obtained when two rabbits were injected with the human DJ5antigen conjugated to keyhole limpet hemocyanin. As can be seen in FIG.2, the amino acid sequences corresponding to DJ5 for rabbit and humanGDF8 are identical, whereas the amino acid sequence of goat DJ5 isdifferent. Therefore, in view of the data provided above for the goat,the preliminary rabbit data suggests that it may be advantageous to usea DJ5 antigen that comprises a different amino acid sequence than thatfor the corresponding region/portion of the host animal GDF8. Thus, inthis case, the ability of the recombinant human GDF8 prohormone toinduce bio-neutralizing antibodies in a goat may be due, at least inpart, to the fact that the antigen used comprised an amino acid sequencethat differs from that of the host sequence by a single amino acidsubstitution in the DJ5 region/portion of GDF8 [see, amino acid residue333 in FIG. 2]. More particularly, as FIG. 2 shows, the Arg₃₃₃ in thehuman sequence is replaced by a Lys₃₃₃ residue in the goat sequence.This lone conservative amino acid substitution may constitute analteration that is significant enough to render the protein “foreign” tothe goat immunological surveillance system.

Example 5 GDF8 Neutralizing Rat mAB 788 Defines A SpecificNeutralization Epitope of the GDF8 Protein

Rat monoclonal antibody 788 is reported to neutralize mouse GDF8bioactivity (R&D Systems Inc., Cat. No. MAB788, Minneapolis, Minn.). Inorder to confirm this result we assayed the monoclonal antibody forneutralization activity against the GDF8 protein. The antibody wascharacterized as described by Example 2, above. The results of thisassay are summarized by Table 5, as follows.

TABLE 5 GDF8 neutralization titers for monoclonal antibody 788 Sample(μg IgG) % Inhibition of GDF8 Activity MAB-788 (12.5) 47 MAB-788 (6.3)17 MAB-788(3.1) 7 MAB-788 (1.8) 0 MAB-788 (0.1) 0

Table 5 confirms that this antibody is capable of neutralizing theactivity of the GDF8 protein. In order to determine the specificity ofthis neutralizing immune response the rat monoclonal antibody wasassayed for its reactivity with a set of seven overlapping peptides(DJ1-7 see table 2 and FIG. 1) that span the entire coding region of theactive GDF8 protein. Reactivity of the monoclonal antibody to eachindividual peptide was determined by ELISA assay (see materials andmethods).

TABLE 6 ELISA reactivity of Rat MAB 788 (10 mg/ml) to GDF8 active regionpeptides OD 405/ minute × 1000 Antigen 1:20 1:40 1:80 DJ1* 4 0 0 DJ2* 00 0 DJ3* 0 0 0 DJ4* 0 0 0 DJ5* 133 118 102 DJ6* 0 0 0 DJ7* 0 0 0proGDF8** 132 127 132 *peptide, **protein

The rat monoclonal antibody reacted specifically with both the purifiedGDF8 prohormone and with the DJ5 peptide. Typically a monoclonalantibody has mono specificity to a single epitope. Among the GDF8 activeregion peptides this monoclonal antibody reacts specifically andexclusively with the DJ5 peptide. This result provides furtherindependent evidence that the DJ5 peptide defines a neutralizing epitopeof the GDF8 protein.

Many modifications and variations of this invention can be made withoutdeparting from its spirit and scope, as will be apparent to thoseskilled in the art. The specific embodiments described herein areoffered by way of example only, and the invention is to be limited onlyby the terms of the appended claims, together with the full scope ofequivalents to which such claims are entitled. Numerous references arecited in the specification, including Genebank accession numbers ofpublished and/or internet-published nucleic acid and polypeptide/proteinsequences, the disclosures of which are incorporated by reference intheir entireties.

1-10. (canceled)
 11. A nucleic acid molecule that encodes a fusionprotein comprising (i) a peptide consisting of 50 or fewer amino acidresidues that comprises amino acid residues 327 to 346 of SEQ ID NO:1and (ii) a heterologous nucleotide sequence.
 12. A nucleic acid moleculethat encodes a peptide consisting of 50 or fewer amino acid residuesthat comprises amino acid residues 327 to 346 of SEQ ID NO:1 comprisingone or more amino acid substitutions; wherein there are no more thanfive amino acid substitutions between amino acid residues 327 to residue346; and wherein the peptide specifically binds to rat monoclonalantibody
 788. 13. A nucleic acid molecule that encodes a peptideconsisting of 50 or fewer amino acid residues that comprises amino acidresidues 327 to 346 of SEQ ID NO:1.
 14. The nucleic acid molecule ofclaim 13 that comprises a nucleic acid sequence of nucleotide 1112 tonucleotide 1171 of SEQ ID NO:
 2. 15-17. (canceled)
 18. The host cell ofclaim 42 that is a eukaryotic cell.
 19. A method of producing a GDF8peptide comprising the steps of culturing the host cell of claim 18, andexpressing the encoded peptide. 20-27. (canceled)
 28. The method ofclaim 19, further comprising the step of recovering the GDF8 peptide.29. The nucleic acid of claim 13, wherein the peptide comprises aminoacid residues 321 to 346 of SEQ ID NO:1.
 30. The nucleic acid of claim29 wherein the peptide comprises amino acid residues 320 to 350 of SEQID NO:1.
 31. The nucleic acid of claim 30 wherein the peptide comprisesamino acid residues 312 to residue 361 of SEQ ID NO:1.
 32. The nucleicacid of claim 13 wherein the peptide comprises a specific neutralizationepitope for an anti-GDF8 antibody.
 33. The nucleic acid of claim 32,wherein the antibody is selected from the group consisting of ratanti-GDF8 monoclonal antibody 788 and an IgG fraction of goat anti-GDF8polygonal antiserum.
 34. The nucleic acid of claim 12 wherein thepeptide comprises amino acid substitutions at a position selected fromthe group consisting of residues 328, 329, 331, 333 and 335, andcombinations thereof, wherein, (a) amino acid residue 328 is His, Leu,or Asn; (b) amino acid residue 329 is Gln or Lys; (c) amino acid residue331 is Asn or Ser; (d) amino acid residue 333 is Arg or Lys; and/or (e)amino acid residue 335 is Ser, Pro, or Thr.
 35. The nucleic acid ofclaim 34 wherein the peptide comprises no more than one amino acidsubstitution between residues 327 to residue 346 of the precursor GDF8.36. A nucleic acid molecule that encodes a fusion protein comprising (i)a peptide consisting of 50 or fewer amino acid residues that comprisesamino acid residues 327 to 346 of SEQ ID NO:1 comprising one or moreamino acid substitutions. wherein there are no more than five amino acidsubstitutions between amino acid residues 327 to residue 346; and (ii) aheterologous nucleotide sequence; wherein the peptide specifically bindsto rat monoclonal antibody
 788. 37. A vector comprising the nucleic acidmolecule of claim
 36. 38. A vector comprising the nucleic acid moleculeof claim
 32. 39. A vector comprising the nucleic acid molecule of claim13.
 40. A vector comprising the nucleic acid molecule of claim
 12. 41. Avector comprising the nucleic acid molecule of claim
 11. 42. A host cellcomprising the vector of claim 39.